1. Field of Invention
The present invention relates to methods for characterization of the structure of molecules and, more particularly, to a method for characterizing the three-dimensional surface structure of proteins and protein complexes employing electrospray mass spectrometric analysis (ES-MS) techniques and computational feedback modeling.
2. Description of the Prior Art
The employment of mass spectrometry for identification of chemical structures, molecular weights, determination of mixtures, and quantitative elemental analysis, based on the application of the mass spectrometer, is a known analytical technique. Mass spectrometry may be used to accurately determine the molecular weights and structural information of organic molecules based on the augmentation pattern of molecular ions formed when the molecule undergoes ionization and fragmentation. The mass of molecules may be measured by ionizing the molecules and measuring their trajectories in response to electric and/or magnetic fields in a vacuum.
Organic molecules having a molecular weight of about a few hundred to a few thousand Daltons are of great medical and commercial interest as they include, for example, polypeptides, proteins, DNA, RNA, oligosaccharides, and other macromolecules such as polymers thereof and other useful polymers. Any organic, organometallic, or other molecule may be analyzed by ES/MS. But of most interest are molecules with molecular weights over about 10,000 Daltons. “Electrospray” ionization is amenable to any type of mass spectrometry, and is therefore of considerable utility. Electrospray mass spectrometry (ES/MS) has more recently been recognized as a significant tool used in the study of proteins and protein complexes. Electrospray ionization as a method of sample introduction for mass spectrometric analysis is also known. Generally, electrospray ionization is a method whereby ions are formed at atmospheric pressure and then introduced into a mass spectrometer using a special interface. In electrospray ionization, a sample solution containing molecules of interest and a solvent is typically pumped through a needle or small conductive tube and into an electrospray interface. An electrical potential of several kilovolts may be applied to the needle for generating a fine spray of charged droplets. The droplets may be sprayed at atmospheric pressure into a desolvation tube or chamber containing a heated gas to vaporize the solvent. Alternatively, the needle may extend into an evacuated chamber, and the sprayed droplets then desolvated in the evacuated chamber. The fine spray of highly charged droplets releases molecular ions as the droplets are desolvated. In either case, ions are focused into a beam, which is accelerated by an electric field gradient, and then analyzed in a mass spectrometer.
Because electrospray ionization occurs directly from solution at atmospheric pressure, the ions formed in this process tend to be strongly solvated. To carry out meaningful mass measurements, it is necessary that any solvent molecules attached to the ions be efficiently removed, that is, the molecules of interest must be “desolvated.” In the prior art, desolvation is achieved in one way by interacting the droplets and solvated ions with a strong countercurrent flow (6−9 1/rn) of a heated gas before the ions enter into the vacuum of the mass analyzer.
The use of such a strong countercurrent gas flow is expensive and difficult to operate because the gas flow rate and the temperature need to be controlled precisely and be optimized for each analyte and solvent system. If proper gas flow and temperature conditions are not attained, it can result in either an incomplete desolvation of the ions or a decrease in sensitivity as ions may be swept away by the gas at high flow rate. To enhance the desolvation process, some have used collisional activation by applying an electrostatic field in a region of reduced pressure between the sampling orifice of the mass analyzer and the skimmer.
Although high speed pumping is commonly incorporated to allow for the direct sampling of electrosprayed ions into the mass analyzer, the detailed method of ion transport from atmospheric pressure to vacuum is different in each case. Thus ion transport has been achieved through a 0.2 mm bore 60 mm long glass capillary tube and skimmer and a 1.0 mm diameter sampling orifice and skimmer.
Techniques involving automated analysis and correction of light or magnetic-based spectral data also are well known in art. For example, Dunkel in U.S. Pat. No. 5,572,125 describes a method and system of using regression analysis to correct spectral data for various types of “noise,” such as signal drift, sample saturation, removal of phase, or shim distortions. This method comprises the steps of providing experimental data to the system, initializing and running a simulation model of the experiment, comparing the simulated results with the experimental data, estimating the unknown parameters, and adjusting the simulated data to fit to the experimental model by employing regression analysis to correct the data in an iterative manner until predetermined criteria are met. However, this method does not disclose or suggest a method by which the three dimensional structures of protein or other large molecules may be determined.
Despite the advances in the sensitivity and resolution of spectrometric data made possible by improved sample introduction, desolvation, and data correction methods, very little attention has focused on the final analysis of spectrometric data in terms of determining the three-dimensional conformation of molecular complexes. Thus, a need exists for an effective method employing ES-MS techniques to characterize the surface structure of a molecule, particularly a protein or protein/small molecule complex. Presently, such a method has not been known.